Analogs of hGH having antagonistic activity, and uses thereof

ABSTRACT

Analogs of hGH having the activity of naturally occurring hGH and a similar amino acid sequence varying from the sequence of natural hGh by the addition of one or more amino acids, e.g. methione or methionine-leucine, to the N-terminus of natural hGH have been produced, recovered and purified. Such analogs may be incorporated into pharmaceutical compositions and administered to a subject to increase the level of hGH in the subject. 
     Analogs of hGH which comprise the amino acid sequence of natural hGH from the N-terminus of which one or more amino acids have been deleted, e.g. Met 14  hGh, have been produced, recovered and purified. Such analogs may be incorporated into pharmaceutical compositions and administered to a subject to lower the level of hGH in the subject. 
     A plasmid has been constructed which directs the expression of an analog of hGH having the amino acid sequence methionine-leucine added to the N-terminus of natural hGH. This plasmid has been introduced into Escherichia coli, the resulting Escherichia coli grown and the analog recovered and purified.

This application is a continuation in part of U.S. Ser. No. 514,188,filed July 15, 1983, the contents of which are hereby incorporated byreference into the present application.

BACKGROUND OF THE INVENTION

One aspect of genetic engineering involves insertion of foreign DNAsequences derived from eukaryotic sources into Escherichia coli or othermicroorganisms. A further refinement concerns inducing the resultingmicroorganisms to produce polypeptides encoded by the foreign DNA.Production of polypeptides can be considered a two-step process, eachstep including numerous substeps. The two steps are transcription andtranslation. To produce a polypeptide efficiently and in quantity bothsteps must be efficient. Transcription is the production of mRNA fromthe gene (DNA). Translation is the production of polypeptide from themRNA.

A critical substep of the transcription process is initiation, i.e., thebinding of RNA polymerase to a promoter-operator region. The sequence ofdeoxyribonucleotide bases which is the promoter region may vary andthereby effect the promoter's relative efficiency which depends on theaffinity of the RNA polymerase for the promoter.

The efficiency of translation is affected by mRNA stability. IncreasedmRNA stability permits improved translation. Although the exactdeterminants of mRNA stability are not precisely known, it is known thatmRNA secondary structure as determined by the sequence of its bases hasa role in stability.

The initial substep of translation involves binding of the ribosome to abase sequence on the mRNA known a the Shine-Dalgarno sequence or theribosomal binding site (RBS). The synthesis of polypeptides begins whenthe ribosome migrates along the mRNA to the AUG start codon fortranslation. Generally these codons are found approximately 10 bases"downstream" from the Shine-Dalgarno site. Factors which increase theefficiency of translation include those which enhance binding of theribosomes to the Shine-Dalgarno site. It has been shown that thesecondary structure of the mRNA in the region of the Shine-Dalgarnosequence and the AUG codon and the distance between the Shine-Dalgarnosequence and the AUG codon each plate a critical role in determining theefficiency of translation are premature termination and attenuation.Efficiency of translation can be improved by removing the attenuationsites.

A difficulty encountered in attempts to produce high amounts ofeukaryotic polypeptides in bacterial cells involves the inability ofcells producing large amounts of mRNA to grow efficiently. Thisdifficulty can be eliminated by preventing transcription by a processknown as repression. In repression genes are switched off due to theaction of protein inhibitor (repressor protein) which preventstranscription by binding to the operator region. After microorganismshave grown to desired cell densities, the repressed genes are activatedby destruction of the repressor or by addition of the molecules known asinducers which overcome the effect of the repressor.

Numerous reports may be found in the literature concerning the cloningof eucaryotic genes in plasmids containing the P_(L) promoter from λbacteriophage. (Bernard, H. V. et al., Gene (1979) 5, 59; Derom, C. etal., Gene (1982) 17, 45; Gheysen, D. et al., Gene (1982) 17, 55;Hedgpeth, J. et al., Mol. Gen. Genet. (1978) 163, 197; Remaut, E. etal., (1981) Gene 15, 81; and Derynck, R., et al., Nature (1980) 287,193. In addition, European patent application No. 041.767, publishedDec. 16, 1981 describes expression vectors containing the P_(L) promoterfrom λ bacteriophage. However, none of these references describe the useof the C_(II) ribosomal binding site.

The use of a vector containing the P_(L) promoter from λ bacteriophageand the C_(II) ribosomal binding site has been described. (Oppenheim, A.B. et al., J. Mol. Biol. (1982) 158, 327 and Shimatake, H. andRosenberg, M., Nature (1981) 292, 128.) These publications describe theproduction of increased levels of C_(II) protein but do not involve ordescribe the production of eucaryotic proteins.

In 1982 Shatzman and Rosenberg presented a poster at the 14th MiamiWinter Symposium (Shatzman, A. R. and Rosenberg, M., 14 Miami WinterSymposium, abstract p98 [1982]). This abstract provides a non-enablingdisclosure of the use of a vector containing P_(L) from λ bacteriophage,Nut and the C_(II) ribosomal binding site to synthesize a "eucaryotic"polypeptide (SV40 small T antigen is actually not a eucaryoticpolypeptide but a viral protein) in an amount greater than 5% of thecell protein in an unnamed bacterial host. The operator used is notdefined. Neither an origin of replication nor a gene for a selectablephenotype is identified. This system with which the vector is used isdescribed as including certain host lysogens into which the vector canbe stably transformed. The present invention in one embodiment, i.e.,pMG100, may have certain similarities to this vector. However, it is nottransformed into a host lysogen, but rather into suitable E. coli hoststrains which contain the thermolabile repressor C_(I) and the N genebut from which the rest of the lysogen has been removed. Moreover, ithas been employed to produce bGH and hGH analogs in amounts in excess of20% to total cell protein.

In addition, in other embodiments of this invention ribosomal bindingsites which differ from C_(II) are employed. Also, in the presently mostpreferred vectors, pND5 and its derivatives, nonessential sequences havebeen removed to create a vector permitting polypeptide production inamounts which are more than 10% greater than those obtained with pMG100.

Recently, applicants have learned of the existence of a pending M.Rosenberg U.S. patent application Ser. No. 457,352 by the NationalInstitutes of Health, Dept. of Health and Human Services, U.S.A.Portions of this application have been obtained from the NationalTechnical Information Service, U.S. Dept. of Commerce. However, theclaims are not available and are maintained in confidence. The availableportions of the application have been reviewed. This disclosure is notenabling. It indicates that the host is important (p8, line 17) butfails to identify any suitable host. It further depends upon the use ofa λ mutant which is not specified (p4, line 20). It indicates that thehost contains lysogens (p8, line 18) unlike the present invention inwhich the host is not lysogenic. It mentions cloning and expression of aeucaryotic gene, monkey metallothionein gene, (p7, line 18) but does notprovide details. It specifies that neither the sequence nor the positionof any nucleotide in the C_(II) ribosomal inding region has beenaltered. (p3, line 27) In the present invention such alteration ispossible.

No disclosure is present in the art concerning: successful expressionwith a P_(L) -C_(II) containing vector system of bovine or human growthhormones; production of bGH or hGH analogs having biological activity;compositions containing such analogs or uses of them; or inductionmethods for achieving polypeptide production in amounts greater than 20%of the total protein produced by the host.

The only disclosure in the art concerning production of bGH analogs byhosts transformed with genetically engineered vectors involves the useof the Trp promoter to produce a bGH analog having the amino acid Met atthe N-terminus of the phenylalanine form of natural bGH (Seeburg, P. H.et al., DNA (1983) 2, 37.

The only disclosure in the art concerning production of hGH analogs byhosts transformed with genetically engineered vectors involves the useof the Lac and Trps promoters to produce an analog of hGH having theamino acid Met at the N-terminus of the natural hGH. (Goedell, D. V. etal., Nature (1979) 281, 544)

SUMMARY OF THE INVENTION

Analogs of hGH having the activity of naturally occurring hGH and asimilar amino acid sequence varying from the sequence of natural hGH bythe addition of one or more amino acids, e.g. methione ormethionineleucine, to the N-terminus of natural hGH have been produced,recovered and purified. Such analogs may be incorporated intopharmaceutical compositions and administered to a subject to increasethe level of hGH in the subject.

Analogs of hGH which comprise the amino acid sequence of natural hGHfrom the N-terminus of which one or more amino acids have been deleted,e.g. Met¹⁴ hGH, have been produced, recovered and purified. Such analogsmay be incorporated into pharmaceutical compositions and administered toa subject to lower the level of hGH in the subject.

A plasmid has been constructed which directs the expression of an analogof hGH having the amino acid sequence methionine-leucine added to theN-terminus of natural hGH. This plasmid has been introduced intoEscherichia coli, the resulting Escherichia coli grown and the analogrecovered and purified.

DESCRIPTION OF THE FIGURES

FIG. 1 Construction of pMH100 expression vector. This plasmid was builtby inserting a fragment of λ phage DNA contained between restrictionsites HaeIII (location 38150) and Sau3a (location 38362) into a pKC30plasmid DNA cleaved with Hpal and BamHl. The HaeIII-Sau3a fragmentcarries nut_(R), t_(R1), cy⁻ and ribosomal binding site of C_(II)protein (C_(II) -RBS). Subcloning of the C_(II) -RBS containing DNA intopKC30 creates pMG100 which contains a unique BamH1 restriction siteright after the ATG initiation codon of C_(II) -RBS and an Nde1restriction site within the ATG triplet (bottom inset). Numbers inparentheses denote location of restriction sites on the λ phage DNA.

FIG. 2. Construction of pRec 2/3 plasmid. A bGH cDNA containing plasmid,D₄, was digested with HaeII. A resulting 1600 bp large fragment waspurified and subjected to digestion at 37° C. for 5 minutes with 5 unitsof S1 exonuclease. A synthetic EcoR1 linker with the sequence:

    GGAATTCC

    CCTTAAGG

was attached by ligation. The product was cleaved with EcoR1 andinserted into pBR322 which has been cleaved with EcoR1. A clone, pALR1,was isolated which upon cleavage with EcoR1 released a 1200 bp fragmentwith the sequence: ##STR1## at the 5' end. Formation of this sequencedemonstrates that pALR1 contains an EcoR1 restriction site whichincludes the TTC codon for residue number 1 (phenylalanine) of authenticbGH. pALR1 was subjected to a partial cleavage with Pst1. The digest wasligated with HindIII linkers and cleaved with EcoR1 and HindIII. Thefragment containing bGH cDNA was isolated and subcloned into pBR322between EcoR1 and HindIII restriction sites to give pAL500. Thesubcloned bGH cDNA fragment was then excised from pAL500 with EcoR1 andHindIII, "filled in" with DNA polymerase "Klenow" fragment and insertedinto the pMG100 expression vector (FIG. 1) opened at the BamH1 site andalso "filled in" as above. The resulting vector pREC 2/2, expresses amodified bGH which is altered at its amino terminus as follows:

    MetAspG1nPhe.sup.1 Pro.sup.2 . . . bGH

The plasmid pREC 2/2 was disgested with Pst1 and the fragment containingthe P_(L) promoter and the 5' end of the bGH gene (designated fragmentA) was isolated. This fragment was ligated to a Pst1 fragment from pAL500 (designated fragment B). The then resulting vector, pRec 2/3,expresses a modified bGH which is altered at its amino terminus asfollows:

    MetAspG1nPhe.sup.1 Pro.sup.2 . . . bGH

FIG. 3. Construction of expression vectors pND5, pND55 and pRO11. Aplasmid pOG7 (A. Oppenheim, S. Gottesman and M. Gottesman, J. Mol. Biol.(1982) 158, 327) was cleaved with Nde1. The ends of the large fragmentcarrying the P_(L) promoter nut_(L), t_(R) and C_(II) -RBS were ligatedto give the pND5 expression vector. This pND5 vector DNA is opened withNde1. Insertion of that Nde1 fragment from pRec 2/3 (FIG. 2) whichcontains bGH cDNA results in a plasmid pRO11 which appears to be abetter expressor of the modified bGH described in FIG. 2 than pRec 2/3.Insertion of synthetic linkers with the sequence: ##STR2## into pOG7cleaved with Nde1 results in a expression vector pND55 which contains aunique Sac1 restriction site in front of ATG. When pND55 is cleaved withSac1 and treated with DNA polymerase "Klenow" fragment an ATG initiationcodon results which follows the P_(L) promoter and C_(II) -RBS. Thisvector is suitable for expression of a wide variety of eukaryotic geneslacking an ATG initiation codon.

FIG. 4. Construction of pTV 18(1) and pTV 104(2). A plasmid, pTVHGH wasprepared by cloning cDNA encoding hGH into the HindIII site of pBR 322using standard methods. Meth. Enzymol. (1979) 68, 75. This plasmid wasdigested with HindIII. The resulting 800 base pair fragment was purifiedand further digested with FnuDII and "filled in" with DNA polymerase"Klenow" fragment. This treatment removes codons for the first 16 aminoacids of hGH. The resulting DNA fragment is ligated with a syntheticlinker which restores the codons for the sequence of hGH from Met¹⁴ andregenerates an Nde1 restriction site in front of the ATG codon forMet¹⁴. After treatment with Nde1 this semisynthetic DNA was insertedinto the pND5 vector opened with Nde1. The resulting plasmid pTV 18(1)(formerly designated pTV 1(8)) expresses hGH under control of the P_(L)promoter. This hGH is an analog missing the first 13 amino acid residuesand having at its N-terminus Met¹⁴.

Plasmid pTV 18(1) was partially digested with Nde1 and ligated with asynthetic linker which contains the codons for amino acids 1-13 of hGH:##STR3##

The linker is also complementary to the Nde1 site on pTV 18(1) andpositions the complete hGH gene in phase with the ATG initiation codonof the pND5 expression vector (FIG. 3). Thus, the resulting plasmid, pTV104(2), expresses native hGH with an extra methionine at the N-terminus.

FIG. 5 shows the vector pAL Trp 46 which contains the Trp promoter andthe first seven amino acids of the Trp E gene transcriptionally fused tothe β-galactosidase gene.

FIGS. 6, 7 and 8 show a series of expression vectors (Tac) containing apart of the Trp promoter and Lac operator followed by restriction sitesfor insertion of a desired gene and expression of bGH under the controlof Tac promoter.

FIGS. 9 and 10 show expression vectors containing bGH cDNA under thecontrol of the histidine promoter.

FIG. 11 shows insertion of the bGH gene into an expression vector underthe control of the Lac promoter.

FIG. 12 shows expression of bGH gene under control of Omp F promoter.

FIG. 13. Construction of Met⁴ -bGH analog pAL401 and expression vectorspND6 and pND11 with altered restriction sites: pAL401 which expresses amodified form of bGH which is lacking the first three amino acids at theamino terminus of the bGH (Met⁴ bGH) was constructed by triple ligationof the following:

(a) a bGH DNA fragment of 623 base pairs with PvuII and HindIII excisedfrom pAL500.

(b) a linker formed by synthesizing two DNA strands which afterpurification were annealed to form: ##STR4## which was "filled in" withDNA polymerase "Klenow" fragment and then cleaved with Nde1 and PvuII toprepare a 58 base pair fragment which was recovered and purified.

(c) pND11 which was prepared as follows. An expression vector pOG7 wasaltered by elimination of HindIII and one of the Nde1 sites (distantfrom the ATG initiator codon) to obtain pND6. Then HindIII linkers wereintroduced into a Sal1 site to give pND11.

FIG. 14. Construction of authentic bGH modified with methionine at theamino terminus and various analogs of bGH.

(a) Plasmid pAL401 is treated with Nde1. A synthetic DNA linkercontaining an ATG initiation signal and the code for the first threeamino acids at the amino terminus of native bGH is ligated into the Nde1site. The resulting vector pAL601 leads to the expression of native bGHcontaining an extra methionine residue at the amino terminus.

(b) Using the strategy described in (a) but modifying the structure ofthe oligodeoxyribonucleotide linker a class of vectors coding for aseries of modified bovine growth hormones is constructed. The modifiedgrowth hormones start with methionine at the N-terminus and are followedby any of the twenty naturally occurring amino acids in each ofpositions 1 and 2, and any of the twenty amino acids other than Glu,Gln, Lys, Met or Trp in position 3. Proceeding from position 4 to theCOOH-terminus the sequence is identical to that of native bGH.

FIG. 15. Tibia test. This figure shows the comparison between effect ofpRec 2/3 bGH analog and authentic bGH on the bone plate growth ofhypophysectomized rats. FIG. 16. Construction of pTV 333(31). pTV333(31)(ATCC accession No. 39981) directs the expression of a novel hGH analoghaving the sequence met-leμ-hGH. pTV333(31) was constructed by adding asynthetic oligonucleotide sequence to the 5' terminus of the Met¹⁴ hGHof pTV18(1). (See FIG. 4). The synthetic oligonucleotide sequence addedwas: ##STR5##

FIG. 17. Construction of pTV304(10). pTV304(10) is similar to pTV104(2)in that it directs the expression of Met-hGH.

It differs from pTV104(2) in that the nucleotide sequence at the 5' endof the gene corresponds exactly to that found in the human genome.pTV304(10) was constructed by adding a synthetic oligonucleotidesequence at the 5' end of the hGH gene in the plasmid pTV18(1). (SeeFIG. 4). The sequence of the synthetic oligonucleotide was: ##STR6##

FIGS. 18-23 set forth results obtained in tests of the activity of hGHanalogs produced by pTV18(1) and pTV104(2). The tests are described morefully in Example 9.

DETAILED DESCRIPTION OF THE INVENTION

A vector has been developed which enables the achievement of enhancedlevels of gene expression and polypeptide expression. The vector is adouble-stranded DNA molecule. Upon introduction into a suitablebacterial host cell containing the thermolabile repressor C_(I) andincreasing the temperature of the host to a temperature at which therepressor is destroyed, the vector renders the host cell capable ofeffecting expression of a desired gene inserted into the vector andproduction of polypeptide encoded by the gene.

The vector includes in 5' to 3' order the following:

a DNA sequence which contains the promoter and operator P_(L) O_(L) fromlambda bacteriophage;

the N utilization site for binding antiterminator N protein produced bythe host cell;

a DNA sequence which contains a ribosomal binding site for rendering themRNA of the desired gene capable of binding the ribosomes within thehost cell;

an ATG initiation codon or a DNA sequence which is converted into an ATGinitiation codon upon insertion of the desired gene into the vector; and

a restriction enzyme site for inserting the desired gene into the vectorin phase with the ATG initiation codon.

The vector also includes a DNA sequence which contains an origin ofreplication from a bacterial plasmid capable of autonomous replicationin the host cell and a DNA sequence which contains a gene associatedwith a selectable or identifiable phenotypic trait which is manifestedwhen the vector is present in the host cell.

The host for use with the vector is Escherichia coli. The presentlypreferred strains are A1637, A1645, A2602 and A1563. A1637 is presentlythe most preferred strain. It was obtained from C600 by insertingtransposon containing tetracycline resistance gene within the galactoseoperon as well as the lambda system for expression which is close togalactose operon. It has been deposited with the American Type CultureCollection in Rockville, Maryland, U.S.A. containing various plasmids asdescribed more fully hereinafter. All such deposits were made pursuantto the Budapest Treaty on the International Recognition of the Depositof Microorganisms.

A1645 was obtained from A1637 by selection for Ga1⁺ (ability to fermentgalactose) as well as loss of tetracycline resistance. It still containsthe lambda expression system but part of the transposon has been removedby selection. Its phenotype is C600 r⁻ m⁺ gal⁺ thr⁻ leu⁻ lac Z⁻ (λcI857Δ H1 ΔBAM N+).

A2602 and A1563 are derived from SA500. Their phenotypes are SA500 his⁻ilu⁻ gal⁺ Δ8(λCI857ΔH1ΔBAM N+ and SA500 his⁻ 0 ilu⁻ gal⁺ Δ8 lac Z×A21(ΔCI859 int2×is1 nutL3 ΔH1), respectively.

Preferably the vector is a covalently closed circular double-strandedmolecule. However, it is not essential that the vector be covalentlyclosed.

The vector achieves its enhanced expression levels after the host cellis heated to a temperature at which the C_(I) repressor is destroyed. Atemperature above about 42° C. is effective for this purpose and sinceit is desired that unnecessary heat damage to the host cells be avoidedto as great an extent as possible, it is generally desirable that thetemperature never exceed 42° C. by more than a few degrees.

One important component of the vector is the ribosomal binding site.Suitable sites are C_(II) from lambda bacteriophage having the sequence:

    TAAGGAAATACTTACAT

    ATTCCTTTATGAATGTA;

a synthetic oligonucleotide having the sequence:

    TAAGGAAGTACTTACAT

    ATTCCTTCATGAATGTA;

and

the major head protein gene of bacteriophage lambda having the sequence:

    TTTTTTTACGGGATTTTTTTATG

    AAAAAAATGCCCTAAAAAAATAC.

Another component of the vector is the restriction enzyme site forinsertion of desired genes into the vector in phase with the ATGinitiation codon. Numerous such sites may be used. The presentlypreferred sites are BamH1, Sac1 and Nde1.

The vector also includes an origin of replication from a bacterialplasmid capable of autonomous replication in the host cell. Suitablesuch origins of replication may be obtained from a number of sources.Presently preferred are origins of replication derived from pBR322 orpR1.

A DNA sequence which contains a gene associated with a selectable oridentifiable phenotypic trait which is manifested when the vector ispresent in the host cell is also a component of the vector. Suitablegenes include those associated with temperature sensitivity or drugresistance, e.g., resistance to ampicillin, chloramphenicol ortetracycline.

Relative to vectors previously described in the scientific literature,the vectors of this invention may be used to obtain enhanced expressionof a wide variety of genes encoding desirable polypeptide products.Suitable genes include those encoding growth hormones, e.g., bovine,porcine, chicken or human growth hormones; superoxide dismutase;apoprotein E; viral protein 1 of foot and mouth disease virus, protein Afrom S. aureus, interleukin III, enzymes, or analogs of any of thepreceding. By analog is meant a polypeptide having the same activity asthe naturally occurring polypeptide but having one or more differentamino acids at the N-terminus of the polypeptide.

The vector may be formed by methods well known to those skilled in theart to which the invention relates. Such methods are described ingreater detail in various publications identified herein, the contentsof which are hereby incorporated by reference into the presentdisclosure in order to provide complete information concerning the stateof the art.

One presently preferred vector is pMG100 having the restriction mapshown in FIG. 1. This vector has had cDNA encoding bovine growth hormoneinserted into its BamH1 restriction site. The resulting plasmid isdesignated pRec 2/3 bGH. Its restriction map is shown in FIG. 2. Theplasmid pRec 2/3 bGH was introduced into Escherichia coli strain A1637using conventional transformation methods. The resulting host vectorsystem has been deposited under ATCC No. 39385.

A second presently preferred vector is pND5 having the restriction mapshown in FIG. 3. Bovine growth hormone cDNA has been inserted into itsNde1 restriction site. The resulting plasmid is designated pRO11. Itsrestriction map is also shown in FIG. 3. The plasmid pRO11 wasintroduced into E. coli strain A1637 via transformation. The host vectorsystem which resulted has been deposited under ATCC No. 39390.

The vector pND5 has also been used to clone human growth hormone. Oneplasmid designated pTV 18(1) and another designated pTV 104(2) have beencreated by inserting hGH cDNA into the Nde1 restriction sites. pTV 18(1)is shown in FIG. 4. It has been introduced into E. coli strain A1637 viatransformation. The resulting host vector system has been depositedunder ATCC NO. 39386. pTV 104(2) is shown in FIG. 4. It also has beenintroduced into E. coli strain A1637. The resulting host vector systemhas been deposited under ATCC No. 39384.

Under the same approach other plasmids may be prepared by inserting intothe restriction enzyme site of a vector of the invention a gene encodinga desired polypeptide.

The preceding specific host vector systems involve E. coli A1637.However, as previously indicated other strains have been used includingA1645, A2606 and A1563. These host vector systems may be used to producepolypeptides such as bovine and human growth hormones. To do so the hostvector system is grown under suitable conditions permitting productionof the polypeptide which is then recovered.

Suitable conditions involve growth of the host vector system for anappropriate period of time at about 42° C. followed by continued growthat about 37°-39° C. for an additional period of time, the growth beingcarried out on a suitable medium.

Desirably the initial period of growth is about 10 to 30 minutes at 42°C. followed by growth at 37°-39° C. for a sufficient period of time suchthat the total period of growth is about 60 to 90 minutes. Preferablythe growth is for about 15 minutes at 42° C. followed by about 75minutes at 38°-39° C. Suitable media include lactalbumin hydrolysatewith addition of glucose and brain heart infusion. In order to stablymaintain the vector in the host it is critical that the host bemaintained under selective pressure, e.g., addition of antibiotic.

By means of the preceding method a number of bGH and hGH analogs hasbeen prepared. These have or may have the activity of the naturallyoccurring hormones.

bGH analogs have the activity of natural bGH and an identical amino acidsequence except for variations at the N-terminus of up to five (5)aminoacids. Examples include the following:

(1) amino acid methionine added to N-terminus of the phenylalanine formof bGH.

(2) amino acid methionine added to N-terminus of the alanine form ofbGH.

(3) amino acid sequence Met-Asp-Gln added to N-terminus of thephenylalanine form of bGH.

(4) amino acid sequence Ala-Gly added to N-terminus of the alanine formof bGH.

(5) amino acid sequence Met-Gly added to N-terminus of the alanine formof bGH.

(6) amino acid sequence Met-Asp-Pro-Met-Gly added to N-terminus of thealanine form of bGH.

(7) amino acid sequence Met-Asp-Pro added to N-terminus of thephenylalanine form of bGH.

(8) amino acid sequence Met-Thr-Arg added to N-terminus of thephenylalanine form of bGH.

(9) amino acids up to methionine (4 position) removed from N-terminus ofphenylalanine form of bGH.

An analog of bGH having the amino acid sequence:

    Met-(X).sub.n -Y-Met . . .

wherein Met is the N-terminus, X is any of the twenty naturallyoccurring amino acids, Y is any of the twenty amino acids other thanGlu, Gln, Lys, Met or Trp, n is an integer from 0 to 6 and Met . . . isthe sequence of natural bGH from position 4 to the COOH-terminus(position 191).

hGH analogs have the activity of natural hGH and an identical amino acidsequence except for variations at the N-terminus. Examples include thefollowing:

(1) amino acid methionine added to N-terminus of natural hGH.

(2) amino acid sequence methionine-leucine added to the N-terminus ofnatural hGh.

Each of these variations involve the addition of one or more amino acidsto the N-terminus of natural hGH.

In addition, this invention provides antagonists of hGH activity whichcomprise the amino acid sequence of natural hGH from which one or moreamino acids have been deleted.

One such analog of hGH has the amino acid sequence:

    Met-(X).sub.n -Y-Met . . .

wherein Met is the N-terminus, X is any of the twenty naturallyoccurring amino acids, Y is any of the twenty amino acids other thanGlu, Gln, Lys, Met or Trp, n is an integer from 0 to 13 and Met . . . isthe sequence of natural hGH from position 14 to the COOH-terminus(position 191).

Another such analog has the amino acid sequence of natural hGH fromwhich the first thirteen amino acids have been removed to yield methione(14 position) at the N-terminus followed by amino acids 15-191 ofnatural hGH.

Pharmaceutical compositions may be prepared which contain effectiveamounts of one or more hGH analog having hGH activity and a suitablecarrier. Such carriers are well-known to those skilled in the art. Theanalogs may be administered directly or in the form of a composition toa human subject, e.g., one afflicted by dwarfism, to treat deficienciesin hGH production by the subject.

Similarly, pharmaceutical compositions may be prepared which containeffective amounts of one or more hGH analog having antagonistic hGHactivity and a suitable carrier. Such carriers are well known to thoseskilled in the art. The analogs may be administered directly or in theform of a composition to a human subject, e.g. one producing excess hGH,to treat or reduce such excess hGH production by the subject.

EXAMPLES

The examples which follow are set forth to aid in understanding theinvention by are not intended to, and should not be so construed as to,limit its scope in any way. The examples do not include detaileddescriptions for conventional methods employed in the construction ofvectors, the insertion of genes encoding polypeptides of interest intosuch vectors or the introduction of the resulting plasmids intobacterial hosts. Such methods are well-known to those skilled in the artand are described in numerous publications including the following:

Principles of Gene Manipulation, An Introduction to Genetic Engineering,2nd Edition, edited by R. W. Old and S. B. Primrose, University ofCalifornia Press (1981).

Met. Enzymol. Vol. 68, Recombinant DNA, edited by Ray Wu.

Met. Enzymol. Vol. 65, Nucleic Acids (Part 1), edited by LawrenceGrossman and Kivie Moldave.

T. Maniatis, E. F. Fritsch and J. Sambrook, Molecular Cloning; ALaboratory Manual, Cold Spring Harbor Laboratory, NY (1982).

H. V. Bernard et al., Gene (1979) 5, 59.

A. B. Oppenheim et al., J. Mol. Biol. (1982) 158, 327.

E. Remaut et al., Gene (1981) 15, 81.

EXAMPLE 1 EXPRESSION VECTORS

As used herein the term expression vector refers to a group of plasmidsuseful for expressing desired genes in bacteria, particularly in E.coli. The desired gene may be inserted into the expression vector oralternatively, the promoters on the expression vector may be excised andplaced in front of the desired gene.

I. P_(l) Expression Vectors

A. pMG 100

pMG 100, as shown in FIG. 1 and described in detail under Description ofthe Figures is composed of λ DNA inserted into a multicopy plasmidpBR322. The salient features of the λ DNA is that it contains the λP_(L) promoter, N utilization sites L and R (nut_(L) and nut_(R))termination R1 site (t_(R1)), the C_(II) ribosomal binding site and anATG initiation codon. Other features are shown in FIG. 1.

pMG100 was prepared form pKC30. pKC30 in turn was prepared by subcloningof λ P_(L) promoter in the following manner.

λ phage DNA was digested with Xho1 and Sma1 restriction endonucleasesand the unique fragment comprised of 6393 base pairs was purified andsubsequently digested with HindIII and BamH1 restriction endonucleases.The resulting fragment comprised of 2397 base pairs and containing P_(L)promoter was purified and ligated into a pBR322 DNA large fragmentisolated from the HindIII and BamH1 digest. The subclone was identifiedby colony hybridization, recovered and plasmid DNA isolated (Oppenheim,A. et al., J.Mol.Biol. (1982) 158, 327).

This plasmid and its derivatives containing eukaryotic genes may bemaintained in suitable E. coli hosts. The most important feature of thehost is that it provides the thermosensitive repressor CI857 and theantitermination N protein. (Gottesman, M. E. et al., J.Mol.Biol. (1978)140, 197).

This vector has numerous advantages over previously described expressionvectors including:

1. Extremely High Levels of Expression: This vector is capable ofdirecting expression of foreign proteins in E. coli at levels as high as15-25% of the total cellular protein.

2. Thermoinducible Regulation of Expression: The P_(L) promoter isinactive when the CI repressor is bound to it. The CI857 repressor isthermosensitive, that is, it binds to the promoter at 30° C. but isinactivated at 42° C. Thus, by increasing the temperature offermentation to 42° C. the host bacteria are induced to produce thedesired protein.

The advantages of such a system include the following:

(a) a foreign protein which is toxic to E. coli can be produced whendesired thus avoiding cell death early in the fermentation process.

(b) overproduction of a protein may stabilize it and prevent proteolyticdegradation. (Cheng, Y. E. et al., Gene (1981) 14, 121) Thus,"instantaneous" overproduction using a tightly regulated promoter suchas P_(L) may be preferable to continuous low level production.

3. High Copy Number: The P_(L) promoter in pMG100 is found on a plasmidwith a high copy number in distinction to λ itself which is present inlow copy numbers in E. coli. This increases expression levels.

4. Ribosome Binding Site and Initiation Codon: This expression vectorcontains a strong procaryotic ribosomal binding site (RBS) as well as atranslation initiation codon (ATG). Thus, any eukaryotic gene may becloned without the need for adding an initiation codon. Furthermore, theefficient RBS increases levels of expression.

5. Convenient Restriction Site: The expression vector has a BamHI sitelocated directly following the ATG initiation codon which permits properpositioning of the desired gene in order to achieve optimal expression.

6. Nut Site; N protein which is provided by the host binds to Nut siteon the expression vector and thereby prevents termination oftranscription at the t_(R1) site.

B. pND5

As shown in Fig. 3, pND5 contains the P_(L) promoter and the otherimportant components of the expression vectors of this invention. Itincludes a unique Nde1 site immediately after the ribosomal bindingsite. The ribosomal binding site differs from the normal C_(II) site. Ithas the sequence:

    TAAGGAAGTACTTACAT

    ATTCCTTCATGAATGTA

It may be derived from a mutant or may be chemically synthesized. Asdescribed in detail under Description of the Figures pND5 was derivedfrom pOG7. (Oppenheim, A., et. al., J.Mol.Biol. (1982) 158, 327) Thisvector does not contain a translation initiation codon. It appears toprovide superior expression of modified bGH and hGH, particularlyenhanced yield relative to pMG100 containing a bGH analog.

C. pND55

pND55 is a derivative of pND5 which contains the convenient restrictionsite Sac1 in front of C_(II) -RBS and ATG initiation codon. Cleavage ofthe plasmid at this site and subsequent treatment with DNA polymeraseKlenow fragment allows one to obtain an ATG initiation codon to whichany desired gene can be ligated. (FIG. 3 and Description of FIG. 3.)

II. Trp Expression Vectors

A. pAL Trp 46

pAL Trp 46 contains the Trp promoter and the first seven amino acids ofthe Trp E gene fused to the β-galactosidase gene. (FIG. 5). The desiredgene can be inserted into a BamHI site which follows the 7 amino acidsof Trp E.

B. pAL Trp 47; Trp 46 Deleted of Attenuator

This is a construction based on Trp 46 in which the attenuator region ofthe Trp promoter has been deleted.

C. Trp-Lac Fusions

The construction of this promoter, found on plasmid p4754 is illustratedin FIGS. 6 and 7. A variation of this construction is outlined in FIG.8.

III. Histidine Promoter Expression Vectors

The construction of this expression vector is illustrated in FIGS. 9 and10.

IV. Other Promoters Used

A. Lac

This promoter was used in the construction of pYL 301 as shown in FIG.11.

B. Omp F

This is a promoter system which expresses a protein attached to a signalsequence. The signal sequence is removed when the protein istranslocated across the membrane. (FIG. 12)

EXAMPLE 2 Bovine Growth Hormone

The starting point for bGH cDNA modifications is plasmid D₄ which hasbeen described previously. (Keshet, E. et al, Nucleic Acids Research(1981) 9, 19). The D₄ plasmid is also described in pending U.S. patentapplication, Ser. No. 245,943, filed Mar. 20, 1981, claiming priority ofIsrael patent application, Ser. No. 59,690 filed Mar. 24, 1980. It haspreviously been deposited with the American Type Culture Collection inan E. coli host under ATCC No. 31826.

I. pRec 2/3 bGH

The construction of pRec 2/3 is shown in FIG. 2 and described in theDescription of the Figures. bGH cDNA from D₄ has been manipulated priorto insertion into PMG100 to provide the correct reading frame.

pRec 2/3 has been introduced into various E. coli strains includingA1637 by transformation using known methods. A1637 containing pRec 2/3has been deposited under ATCC No. 39385. This strain produces upongrowth and induction an analog of bGH having the amino acid sequenceMet-Asp-Gln added to the N-terminus of the phenylalanine form of naturalbGH. The amount of bGH analog produced by pRec 2/3 was about 23% of thetotal protein produced by the bacteria as calculated from scanning ofCoomasie stained SDS polyacrylamide gels.

pRO11

The construction of pRO11 is shown in FIG. 3 and described in theDescription of the Figures. The pND5 vector DNA is restricted with Nde1.Insertion of the Nde1 fragment from pRec 2/3 (FIG. 2) which contains bGHcDNA results in the plasmid pRO11.

pRO11 has been introduced into E. coli A1637 by transformation. Theresulting host vector system has been deposited under ATCC No. 39390.This strain was grown and induced produces the same analog as pRec 2/3.Preliminary results indicate that pRO11 produces up to 20% more bGHanalog than pRec 2/3. The methods used to grow the strain, recover thebGH analog produced and purify it are the same as those described forpRec 2/3 in Example 4.

III. pAL401

The construction of pAL401 is shown in FIG. 13 and described in theDescription of the Figures. bGH cDNA from D₄ by way of pAL-500 (FIG. 2)was inserted into pND11 as shown in FIG. 13.

pAL401 may be introduced into E. coli A1637 by transformation. Theresulting strain produces an analog of bGH in which Met⁴ of natural bGHis at the N-terminus and the amino acids preceding Met⁴ have beendeleted.

IV. pAL601

The construction of pAL601 is shown in FIG. 14 and described in theDescription of the Figures. It is a derivative of pAL401 (FIG. 13).

pAL601 may be introduced into E. coli A1637 by transformation. Theresulting strain produces an analog of bGH in which Met has been addedto the N-terminus of the phenylalanine form of bGH.

EXAMPLE 3 Human Growth Hormone

The starting point for hGH cDNA was cloning of the cDNA from mRNApurified from hypophyses tumor from acromegalic patients into theHindIII site of pBR322.

I. pTV 18(1)

The construction of PTV 18(1) is shown in FIG. 4 and described in theDescription of the Figures. hGH cDNA was manipulated prior to insertioninto pND5 to provide the correct reading frame.

pTV 18(1) was introduced into E. coli A1637 by transformation. Theresulting bacteria have been deposited under ATCC No. 39386. This strainupon growth and induction produces an analog of hGH having the sequenceof natural hGH beginning with Met¹⁴ and lacking amino acids 1-13. Theamount of hGH analog produced by pTV 18(1) was about 8% of the totalprotein produced by the bacteria.

II. pTV 104(2)

The construction of pTV 104(2) is shown in FIG. 4 and described in theDescription of the Figures. hGH cDNA was manipulated prior to insertioninto pND5 to provide the correct reading frame.

pTV 104(2) was introduced into E. coli A1637 by transformation. Theresulting bacteria have been deposited under ATCC No. 39384. This strainupon growth and induction produces an analog of hGH having the sequenceof natural hGH preceded by Met at the N-terminus. The amount of hGHanalog produced by pTV 104(2) was above 25% of the total proteinproduced by the bacteria.

EXAMPLE 4 Growth of pRec 2/3

Stock Cultures: Stock cultures of pRec 2/3 in A1637 are grown on BHImedium (see inoculum), then diluted twofold with 87% glycerol containingphosphate citrate buffer, and stored at -70° C.

Inoculum: Inoculum is propagated in BHI medium (37 g/l) brain heartinfusion (DIFCO). Sterile medium in shake flask is inoculated from stockculture and incubated 15 hours on shaker at 30° C., 200 r.p.m.Subsequent stages in inoculum propagation are carried out in stirredaerated fermentors. Sterile medium is inoculated with 0.2 ml flaskculture per l, and incubated 15 hours at 30° C., pH 7±0.5 with agitationand aeration to maintain dissolved oxygen level above 20% airsaturation.

Production: Production medium contains:

    ______________________________________                                        Lactalbumin hydrolysate                                                       (enzymatic)            20    g/l                                              Yeast extract          10    g/l                                              K.sub.2 HPO.sub.4      2.5   g/l                                              NaCl                   10    g/l                                              Ampicillin             0.1   g/l                                              Biotin                 0.1   mg/l                                             Thiamine               1     mg/l                                             Trace elements solution                                                                              3     ml/l                                             ______________________________________                                    

Ampicillin, biotin and thiamine in solution are filter sterilizedseparately and added to the sterile production medium beforeinoculation. Sterile glucose solution is added initially to supply 10g/l, and during the induction and expression procedure to maintainglucose above 10 g/l

Trace elements solution contains:

    ______________________________________                                        MgSO.sub.4 ·7H.sub.2 O                                                                    170    g/l                                               FcCl.sub.3           16     g/l                                               ZnCl.sub.2 ·4H.sub.2 O                                                                    2      g/l                                               CoCl.sub.2 ·6H.sub.2 O                                                                    2      g/l                                               Na.sub.2 MoO.sub.4 ·2H.sub.2 O                                                            2      g/l                                               CaCl.sub.2 ·2H.sub.2 O                                                                    1      g/l                                               CuCl.sub.2           1      g/l                                               H.sub.3 BO.sub.3     0.5    g/l                                               Conc. HCl            100    ml/l                                              ______________________________________                                    

The medium is inoculated with 5-10% inoculum culture and incubated at30° C. Agitation-aeration rates are set to maintain dissolved oxygenlevel above 20% air saturation. The pH is maintained at 7±0.2 with NH₃.Once cell concentration reaches about 3 g/l (OD₆₆₀ =10) induction isstarted.

Temperature is raised to 42° C. Maintained there for 15 minutes, thenlowered to 38° C. Following incubation at 38° C. for 1-11/2 hours, theculture is chilled, and cells are recovered by centrifugation forhormone purification.

Recovery of bGH

One kilogram of bacterial cells is suspended in 10 volumes of thesolution containing 50 mM Tris-Cl (pH 7.4), 50 mM EDTA and 25% sucrosein a Warring blender, with a control of blender's speed to minimizefoaming. The homogeneous suspension is continuously passed through aDynomill cell disruptor (Willy A. Bachofen, Basel) and the homogeneoussuspension of disrupted cells is clarified first by centrifugation in aSharpless centrifuge followed by a continuous centrifugation at 20,000rpm in a Sorvall centrifuge. The precipitate from both centrifugationsteps is collected, washed with 50 mM Tris-Cl (pH 7.4) and resuspendedin 500 ml of the same buffer. Lysozyme is added to a final concentrationof 2 mg/ml and the suspension is incubated for 1 hour at 37° C. TritonX-100 is then added to a final concentration of 1%, the suspension iscooled to 4° C. and centrifuged at 20,000 rpm for 20 minutes in aSorvall SS34 rotor. The precipitate is collected, washed twice with 50mM Tris-Cl, resuspended in 500 ml of 50 mM Tris-Cl (pH 7.4),5 mM MgCl₂and deoxyribonuclease is added to a final concentration of 20 μg/ml.After incubation for 30 minutes at room temperature the precipitate iscollected as above, washed twice with 500 ml of 20 mM Tris-Cl (pH 7.4),100 mM NaCl and 10 mM EDTA, followed by two washings with 500 ml ofdistilled water. The precipitate is collected by centrifugation and canbe stored at -20° C. for an indefinite time. At this stage the bGH is80% pure as judged by sodium dodecyl sulfate-gel electrophoresis. Theyield is approximately 15 g of bGH.

Purification of bGH

One hundred gr of precipitate is suspended in 40 ml distilled water andsolubilized by titration with 0.5M NaOH, pH 11.8. The solution is thensonicated for 2 minutes and clarified by centrifugation at 20,000 rpm ina Sorvall SS34 rotor for 20 minutes. The solution is then applied onto aSepharose CL-6B column (5×100 cm) equilibrated with 6.5 mM boratebuffer, pH 11.8. Column is developed at the rate of 100 ml/hr andfractions of 12 ml are collected. The first peak off the column isdiscarded. The following two peaks are separated and pooled. The firstrepresents aggregated bGH with low activity; the second bGH with high ADEAE-Sephacel (25 g/100 gr. equiv. ppt) column is equilibrated with 6.5mM borate buffer, pH 9.0. The second bGH peak is brought to pH 9.0 withHCl loaded on the DEAE Sephacel column at a rate of 250 ml/hr. Thecolumn is washed with 7.5 ml of 6.5 mM borate buffer, pH 9.0, elutedwith 6.5 mM borate buffer, pH 9.0 containing 75 mM NaCl. The fractionswith OD₂₈₀ above 0.3 are pooled, dialysed against H₂ O in MilliporePellicon dialysis apparatus and then lyophilized.

EXAMPLE 5 Activity of bGH Analog Produced by pRec 2/3

1. Radioimmunoassay comparison of bGH analog with natural bGH

A solution containing 100 ng/ml bGH analog was prepared in phosphatebuffered saline (1% BSA). This solution was diluted serially toconcentrations of 50, 25, 12.5, 6.25, 3.12, 1.56 and 0.78 ng/l.Duplicate 0.1 ml aliquots of these solutions were submitted to RIA usinga double antibody procedure. The dilution curve was comparable to thatobtained with natural bGH.

2. Radioreceptor binding Assay

A radioreceptor binding assay was performed with rabbit liver membranesas described by T. Tushima and H. G. Freisen (Y. Chin., Endocr. Metab.(1973) 37, 334 using ¹²⁵ I-hGH as the tracer and authentic bGH solutionsfor the construction of calibration curves. Samples were incubated intriplicate for two hours at room temperature in 0.3 ml of assay buffer(50 mM Tris, 15 mM CaCl₂ and 5 mg/ml bovine serum albumin, pH 7.6). Thetubes contained ¹²⁵ I-hGH (20,000 cpm of preparation of 30-60 μci/μg),150-250 μg liver membrane protein and either natural bGH (1-100 ng) orextracts of bacterial bGH. The result demonstrated that the bGH activityof the bGH analog is comparable to that of natural bGH.

3. Tibia Test

The bioactivity of the pRec 2/3 bGH analog recovered from engineeringbacterial cells according to Example 4 was evaluated by a tibia test.(Parlow, A. F., et al., Endocrinology (1965) 77, 1126.) Rats werehypophysectomized at 28-30 days of age, then kept for 10-14 days withouttreatment. Bovine growth hormone derived from bovine pituitaries or fromrecombinant E. coli was dissolved in 0.15M NaCl=0.01M borate, pH 10.0.Rats (4-7 per group) received daily subcutaneous injections of bGHsolutions (5-125 μg/day in 0.2 cc) for 5 days while kept on a normaldiet (Purina Rat-Chow and water ad-libitum). The animals were sacrificedon the 6th day, their foreleg knee-bones taken out, cut longitudinally,fixed with acetone and stained with 2% AgNO₃. The width of theepiphyseal plates were measured by observation through a dissectingbinocular (Nikon). Mean values (of 40 readings per rat) were used forthe construction of log dose-response curves. Results are shown in FIG.15.

EXAMPLE 6 bGH Analogs

Table I sets forth a series of plasmids which have been constructed andthe analogs which were produced from them.

                  TABLE I                                                         ______________________________________                                        PLASMID  AMINO TERMINUS OF bGH ANALOGS                                        ______________________________________                                        Rec 2/3  Met Asp Gln Phe.sup.2                                                pB 1     Met Asp Pro Met Gly Ala Phe.sup.2                                    pM 4     Met Asp Pro Phe.sup.2                                                pM 1     Met Ala.sup.1 Phe.sup.2                                              pM 2     Met Ala.sup.1 Phe.sup.2                                              pAL 401  Met.sup.4                                                            pYL 301  Met Gly Ala.sup.1 Phe.sup.2                                          pAL 302  11 A.A + Ala.sup.1 Phe.sup.2                                         pHis 129 Met Thr Arg Phe.sup.2                                                pAL 312  Met Gly Ala.sup.1 Phe.sup.2                                          pAL 322  Met Gly Ala.sup.1 Phe.sup.2                                          pAL 601R Met Gly Ala.sup.1 Phe.sup.2                                          p 18     Met Gly Ala.sup.1 Phe.sup.2                                          PBTG-800 Met Glu Phe.sup.2                                                    pORF 2-12                                                                              Ala Gly Ala.sup.1 Phe.sup.2                                          ______________________________________                                    

EXAMPLE 7 Effect of pRec 2/3 bGH analog on Lactogenesis in Dairy Cows

The lactogenic effect of bGH has been well documented in the scientificliterature in the reports of Bines, J. et al, Brit J. Nutri. (1980) 43,179 and Peel, C. et al, J. Nutr. (1981) 111, 1662. Bauman, D. et al, J.Dairy Sci. Vol. Supp. 1, Abst 86 (1982) reported that milk productionwas increased by rDNA bGH. An experiment was conducted to determine theeffects of pRec 2/3 bGH on lactogenesis in comparison with natural bGH.Eighteen Holstein cows ranging from 141 to 154 days postpartum wererandomly assigned to treatment and blocked according to milk productionaccording to the following design.

    ______________________________________                                        Pretreatment                                                                             Treatment    Daily GH Injection                                    ______________________________________                                        Control    5 days       Saline                                                Natural bGH                                                                              5 days       25 mg/day for 10 days                                 pRec 2/3 bGH                                                                             5 days       25 mg/day for 10 days                                 ______________________________________                                    

The bGHs were put in solution with 0.1M NaHCO₃ aqueous buffer (pH=8.2)at the concentration of 1 mg/ml immediately prior to each day'sinjections. The cows were injected with placebo or bGH solution dailyfor 10 days in a subcutaneous site in the neck region. No injectionswere given during the 5-day pretreatment period.

The cows were milked twice daily at approximately 6:00 a.m. and 5:00p.m. Milk weights were recorded by the Boumatic system and recorded inthe dairy data system.

The average milk production values for the pretreatment and bGHtreatment periods are shown in Table II. The production level of thecontrol cows was unchanged while the milk volume increased to a similardegree in both the bGH groups. The natural bGH caused an 11.9% increasein milk for a 10-day period and bGH analog treatment resulted in a 10.2%increase. The data were not analyzed for statistical significance due tothe small number of animals, however, the magnitudes of the increasesare similar to those reported in the literature.

It was concluded that pRec 2/3bGH stimulates lactogenesis in dairy cowssimilar to natural bGH.

                  TABLE II                                                        ______________________________________                                        Bovine Growth Hormone Effect on Lactogenesis                                  Natural bGH vs pRec 2/3 bGH                                                            Av. Daily Milk Production                                                     lb/day                                                               Treatment      Pretreatment                                                                             During GH                                                                             % Increase Over                             Group   No.    5 days     10 days Pretreatment                                ______________________________________                                        Control 6      57.23      57.26   --                                          Natural 5      58.54      65.50   11.9                                        bGH                                                                           25 mg/day                                                                     pRec 2/3                                                                              6      57.48      63.34   10.2                                        bGH                                                                           25 mg/day                                                                     ______________________________________                                    

Each cow was injected daily subcutaneously will either placebo or bGHsolution once daily for 10 days.

EXAMPLE 8 Human Growth Hormone Analog

The construction of pTV333(31) (ATCC accession No. 39981) is shown inFIG. 16 and described in the Description of the Figures. Theintermediate plasmids in the construction of pTV333(31) are shown inFIG. 4 and described in the Description of the Figures.

pTV333(31) was introduced into E. coli strain A2097 by transformation.This strain produces upon growth and induction an analog of human growthhormone (hGH) having the amino acid sequence Met-Leu added to theN-terminal amino acid phenylalanine of natural hGH. The amount of hGHanalog produced by this strain was about 6% of the total proteinproduced by the bacteria as calculated by scanning the hGH band onCoomassie blue stained SDS polyacrylamide gels and determining the totalprotein by the method of Lowry.

Growth of pTV333(31)

I. Stock Cultures

Stock cultures of pTV333(31) were grown on casein medium (see Inoculum),then diluted two-fold with freezing medium and stored at -80° C.Freezing medium contains per 500 ml:

    ______________________________________                                        K.sub.2 HPO.sub.4     6.3    g                                                KH.sub.2 PO.sub.4     1.8    g                                                Na Citrate            0.45   g                                                MgSO.sub.4 ·7H.sub.2 O                                                                     0.09   g                                                (NH.sub.4).sub.2 SO.sub.4                                                                           0.9    g                                                Glycerol              44.0   g                                                ______________________________________                                    

II. Inoculum

The inoculum was propagated in 20 g/l Tryptone 10, g/l yeast extract and2 NaCl. Sterile medium in a shake flask was inoculated from stockculture and incubated 15 hours on a shaker at 30° C. and approximately200 r.p.m. As needed subsequent stages in inoculum propagation werecarried out in stirred aerated fermenters. Sterile medium was inoculatedwith 2-10% inoculum and incubated 15 hours at 30° C., pH 7±0.5 withagitation and aeration to maintain a dissolved oxygen level above 20%air saturation.

III. Production

The production medium contains:

    ______________________________________                                        Tryptone               20    g/l                                              yeast extract          10    g/l                                              K.sub.2 HPO.sub.4      2.5   g/l                                              MgSO.sub.4 ·7H.sub.2 O                                                                      1     g/l                                              NaCl                   5     g/l                                              Biotin                 0.1   mg/l                                             Thiamine               1     mg/l                                             Trace elements solution                                                                              3     ml/l                                             ______________________________________                                    

The medium also contains 100 mg/liter ampicillin. The ampicillin isoptional for production but is always found in the medium used forgrowing the inoculum.

Biotin, thiamine and ampicillin in concentrated solution were filtersterilized separately and added to the sterile production medium beforeinoculation. Sterile glucose solution was added initially to supply 10g/l. At the induction step another 10 g/l of glucose was added.

The trace elements solution contains:

    ______________________________________                                        FeCl.sub.3           16     g/l                                               ZnCl.sub.2 ·4H.sub.2 O                                                                    2      g/l                                               CoCl.sub.2 ·6H.sub.2 O                                                                    2      g/l                                               Na.sub.2 MoO.sub.4 ·2H.sub.2 O                                                            2      g/l                                               CaCl.sub.2 ·2H.sub.2 O                                                                    1      g/l                                               CuCl.sub.2           1      g/l                                               H.sub.3 BO.sub.3     0.5    g/l                                               Conc. HCl            100    ml/l                                              ______________________________________                                    

The medium is inoculated with 0.5-10% inoculum culture and incubated at30° C. Agitation-aeration rates are set to maintain a dissolved oxygenlevel above 20% air saturation. The pH is maintained at 7±0.2 with NH₃.Once cell concentration reaches about 3.5 g/l (OD₆₆₀ =10) induction isstarted.

The temperature is raised to 42° C. and maintained at 42° C. for 1-5hours. The culture is then chilled and cells are recovered bycentrifugation for hormone purification.

EXAMPLE 9 Activity of hGH Analogs Produced by pTV18(1) and pTV104(2)

I. Radioimmunoassay comparison of hGH analogs with natural hGH

Solutions containing 1000 ng/ml hGH analogs were prepared in phosphatebuffered saline (1% BSA). These solutions were diluted serially toconcentrations shown in FIG. 18. Duplicate 0.1 ml aliquots of thesesolutions were submitted to RIA using a double antibody procedure. Thedilution curves are shown in FIG. 18.

II. Radioreceptor binding assay

A radioreceptor binding assay was performed with rabbit liver membranesas described by T. Tushima and H. G. Freisen [Y. Chin., Endocr. Meta.(1973) 37, 334] using ¹²⁵ I-hGH as the tracer and authentic hGHsolutions for the construction of calibration curves. Samples wereincubated in triplicate for two hours at room temperature 0.3 ml ofassay buffer 50 mM Tris, 15 mM Cacl₂ and 5 mg/ml in bovine serumalbumin, pH 7.6). The tubes contained ¹²⁵ hGH (20,000 cpm of preparationof 30-60 μci/ug), 150-250 ug liver membrane protein and either naturalhGH (1-1,000 ng) or extracts of bacterial hGH analogs. The results shownin FIG. 19 demonstrated that the hGH activity of the Met-hGH analog iscomparable to that of natural hGH.

III. Tibia and Weight gain test

The bioactivity of the hGH analogs recovered from genetically engineeredbacterial cells was evaluated by a tibia test. [Parlow, A. F., et al.,Endocrinology (1965) 77, 1126]. Rats were hypophysectomized at 28-30days of age, then kept for 10-14 days without treatment. Human growthhormone analogs derived from recombinant Escherichia coli was dissolvedin 0.15M NaCl=0.01M borate, pH 10.0. Rats (4-7 per group) received dailysubcutaneous injections of bGH solutions in 0.2 cc for five days whilekept on a normal diets (Purina Rat Chow and water ad-libitum).

The rats were weighed at the start of the experiment and before beingsacrificed. FIGS. 20 and 21 show the increases in total body weight as afunction of the growth hormone concentration in the daily injection.

The animals were sacrificed on the sixth day, their foreleg knee bonestaken out, cut longitudinally, fixed with acetone and stained with 2%AgNO₃. The width of the epiphyseal plates were measured by observationthrough a dissecting binocular (Nikon). Mean values (of 40 readings perrat) were used for the construction of log dose-response curves. Resultsare shown in FIGS. 22 and 23.

Purification of hGH Analogs

One hundred g of precipitate is suspended in 40 ml distilled water andsolubilized by titration with 0.5M NaOH to pH 10.0. The solution wascentrifuged (20,000 rpm in a Sorvall SS34 rotor for 20 minutes.) Thesupernatant was further titrated to pH 11.5 and then sonicated for twominutes and clarified by centrifugation at 20,000 rpm in Sorvall SS34rotor for 20 minutes. The solution is then applied onto a SepharoseCL-6B column (5×100 cm) equilibratd with 6.5 mM borate buffer, pH 11.5.The column is developed at the rate of 100 ml/hr and fractions of 12 mlare collected. The first peak off the column is discarded. The followingtwo peaks are separated and pooled. The first represents aggregated hGHwith low activity; the second hGH with high activity.

A DEAE-Sephacel (25 g/100 g equiv. ppt) column is equilibrated with 6.5mM borate buffer, pH 9.8. The second hGH peak is brought to pH 9.8 withHCl loaded on the DEAE Sephacel column at a rate of 250 ml/hr. TheColumn is washed with 7.5 ml of 6.5 mM borate buffer, pH 9.8 containing100 mM NaCl. The fractions with OD₂₈₀ above 0.3 are pooled, dialysedagainst H₂ O in Millipore Pellicon dialysis apparatus and thenlyophilized.

EXAMPLE 10 Activity of hGH Analog Produced by pTV18(1)

Lactogenic properties of human growth hormone were described over 20years ago (1) and were confirmed in several species including mice (2)parimates (3) and cows (4). One of the most specific, accurate andsensitive bioassays for lactogenic hormones is based on its mitogeniceffect on NB2 rat lymphoma cells. These cells are absolutely dependenton PRL, hGH or other lactogenic hormones for their proliferation whencultured in 10% horse serum supplemented medium (5-7). This assaytherefore was chosen to study the in vitro effect of the new 20Kderivative of hGH (Met¹⁴ hGH, 14-191) obtained by recombinant DNAtechnology. An additional bioassay used in the present study was thestimulation of fat synthesis in explants from bovine lactating mammarytissue which was found (4) to be highly dependent upon the presence ofbPRL, oPRL or hGH.

Materials and Methods

Materials

Human growth hormone (hGH-83-8-29 H 2.2 IU/mg) was prepared in ourlaboratory; Ovine PRL (oPRL; (oPRL; NIH-P-S12, 35 IU/mg) and bovine PRL(bPRL, 86) were obtained from the Hormone Distribution Program, NationalInstitutes of Health, Department of Health and Human Services, U.S.A.Radioiodinated hGH ([¹²⁵ ]I-hGH) was prepared as described earlier (8,9). Carrier-free Na[¹²⁵ ]I and CH₃ [¹⁴ ]CHOOH (56 mCi/mmole) werepurchased from New England Nuclear Corp. (Boston, MA, U.S.A.).Monoclonal antibodies against hGH were prepared (10). Human growthhormone analog lacking 13 amino acids at the amino terminus, beginningwith the amino acid methionine which is naturally located at residue no.14 (Met¹⁴ hGH) was produced in the efficient host-vector systemdescribed herein which expressed this polypeptide at a level of morethan 15% of total bacterial protein. Gram quantities of the hormone werepurified to a high degree (generally 80% purity). 15% NaDodSO₄/polyacrylamide gel electrophoresis in presence of β-mercaptoethanolgave an expected 20K dalton band, and analytical isoelectrofocusingrevealed that the isoelectric point is close to that of native hGH. Whenlypholized and kept at 4° C. or lower, the protein was quite stable,although after a few months of storage low molecular weight productscould be detected even in the absence of mercaptoethanol. Thus thematerial used in this study consisted of the main 20K band (˜80%) andfew low molecular wight bands (˜20%). However, the molecule exhibited amarked degree of instability upon storage in neutral solutions withrapid accumulation of breakdown products. Met¹⁴ hGH was tested forimmunological cross-reactivity with rabbit anti-hGH. It was found to becapable of displacing the [¹²⁵ ]I-hGH but the displacement curve did notparallel that of hGH, indicating only partial cross-reactivity.

Nb2 lymphoma cells culture

Suspension cultures of Nb2 lymphoma cells (Nb2-11C) were maintained in75 cm² tissue culture flasks (Nunc, Kamstrup, Denmark). Cultureconditions were those described by Gout, et al. (5). To stimulatemaximal cell growth, cells were cultured in Fischer's medium (forleukemic mouse cells) containing FCS (10%) HS (10%), 2-mercaptoethanol(10⁻⁴ M), penicillin (50 U/ml) and streptomycin 50 μg/ml), whichproduced a doubling time of approximately 20 h. Stationary cultures wereobtained by removing FCS from the medium 20-24 hr prior to the additionof the hormone. An incubation atmosphere of 5% CO₂ -95% are used forcell culture. Growth of Nb2 cells was quantified according to thebioassay of Tanaka, et al. (6) with minor modifications. Growing cellswere transferred to a non-growth medium (i.e. without FCS) for 20-24 h.Cells were collected by centrifugation (500×g) and resuspended ordirectly diluted in a fresh medium (without FCS) at a concentration of1-2 ×10⁵ cells/ml. Two ml aliquots were pipetted into 35 mm tissuecultures dishes (Nunc, Kamstrup, Denmark). Samples to be assayed forgrowth-promoting or inhibiting activity were added to the dishes in avolume of 0.05-0.10 ml. Following a 3-day incubation period, thecontents of each dish were added to 8 ml Isoton (Fisher Scientific Co.,Pittsburgh, PA) and cell number was determined using a Coulter Counter(Coulter Electronics Inc., Hialeah, FL). Each sample was assayed induplicate.

Bovine lactating mammary gland explants culture

Preparation of explants from bovine lactating mammary tissue was carriedout as described previously and the media were changed every 24 h (11).Fat synthesis was determined by measuring incorporation of CH₃ [¹⁴ ]COOHto the fatty acid fraction for 4 h, after 4 days of culture and aselected hormonal regime (4). Glucose uptake was determined by measuringthe glucose content of the medium prior to and after incubation.

Binding experiments

Binding studies were performed using [¹²⁵ ]I-hGH in intact Nb2 cells(12) or microsomal solubilized fraction prepared from lactating bovinemammary gland (9). The non-specific binding was determined in thepresence of 1 μg/ml and 4 μg/ml of hGH respectively.

Statistical analysis

Statistical analyses were performed by a two-tailed Student's t test forunpaired comparisons. All parametric data were exposed as an average±SEM.

Results

Antagonistic effect of Met¹⁴ hGH on oPRL or hGH stimulated proliferationof Nb2 cells

Simultaneous addition of Met¹⁴ hGH and either oPRL or hGH resulted in anobvious inhibition of the proliferation rate as compared to controlswithout Met¹⁴ hGH. The inhibition was dose-dependent and competitive,that is, it could be reduced by increased levels of oPRL or hGH, whilethe maximal proliferation rate was not affected. The inhibition could betotally abolished by increasing the concentration of the agonist (Table1). Met¹⁴ hGH alone had no effect on cell growth and the number of cellsin cultures containing 1-10000 ng/ml of Met¹⁴ hGH did not differsignificantly from controls without any hormones. In an additionalexperiment, the cells were preincubated for 24 h in the presence of 20μg/ml of Met¹⁴ hGH. Then they were washed extensively and incubated foran additional 3 days in the presence of 0.125 and 1.0 ng hGH/ml. Theincrease in cell numbers was measured every day and no difference in theproliferation rate, as compared to cells preincubated in medium only,was observed. In all experiments, the inclusion of Met¹⁴ hGH in themedium did not affect cell shape or viability as observedmicroscopically.

                  TABLE 1                                                         ______________________________________                                        Effect of excess hGH or oPRL on the growth                                    inhibition of Nb2 lymphoma cells by Met.sup.14 hGH                                   No. of doublings in presence of                                        Hormone                                                                              Met.sup.14 hGH ( g/ml): *                                              added  ng/ml    None      2         10                                        ______________________________________                                        hGH    100      2.25 ± 0.01                                                                          2.29 ± 0.01                                                                          2.31 ± 0.02                                   25       2.29 ± 0.02                                                                          2.29 ± 0.01                                                                          2.30 ± 0.01                                   1        2.20 ± 0.02                                                                          1.91 ± 0.01§                                                                    0.97 ± 0.02§                      oPRL   200      2.22 ± 0.01                                                                          2.23 ± 0.02                                                                          2.21 ± 0.01                                   20       2.22 ± 0.02                                                                          2.24 ± 0.01                                                                          2.09 ± 0.02§                             2        2.20 ± 0.01                                                                          2.06 ± 0.03.sup.                                                                     1.22 ± 0.01§                      ______________________________________                                                                            1                                          *Through 68 h, initial concentration of cells was 150000 cell/ml              .sup.  av ± SEM, n = 2                                                     .sup.± Significantly different from the control without Met.sup.14 hGH     (p < 0.01).                                                                    § as above (p <  0.001).                                           

In order to validate the similarity of Met¹⁴ hGH to the native hormone,growth of Nb2 cells was stimulated by oPRL and inhibited by addition ofMet¹⁴ hGH. Then the inhibitory effect of the latter was abolished bymonoclonal antibodies against hGH. As shown in Table 2, the abolitionwas dose dependent and specific since ascitic fluid containing anti-hIgEantibodies failed to affect the response. It should be noted that twoother anti-hGH monoclonal antibodies also abolished the inhibitoryactivity of Met¹⁴ hGH while anti-hPRL monoclonal antibody was devoid ofthis effect.

Inhibition of PRL stimulated fat synthesis in explants from bovinelactating mammary glands by Met¹⁴ hGH

The results presented in Table 3 clearly indicate that bPRL-stimulatedfat synthesis was drastically inhibited in explants cultured in thepresence of 10-50 μg/ml Met¹⁴ hGH/ml. Moreover, in explants from Cowsno. 1 and 4, Met¹⁴ hGH also significantly inhibited fat synthesis in theabsence of bPRL. Analysis of the data again suggested competitiveinhibition although it was less obvious than in the experiments usingNb2 cells. Glucose uptake, which served as an index of explant viability(4) was not affected by various hormonal regimes including addition ofMet¹⁴ hGH. It should be noted that in the absence of Met¹⁴ hGH additionof bPRL significantly (p<0.05) increased fat synthesis, although inexplants from some cows (2, 3 and 5) the maximal effect was not reachedat 0.2 μg PRL/ml.

Binding studies

Binding studies with intact Nb2 cells or with solubilized microsomalfraction from bovine lactating mammary gland revealed Met¹⁴ hGH to becapable of inhibiting the specific binding of ¹²⁵ I-hGH in both cases.

                                      TABLE 2                                     __________________________________________________________________________    Effect of monoclonal antibodies against hGH on the growth of                  oPRL-stimulated                                                               Nb2 lymphoma cells by Met.sup.14 hGH                                                     No. of doublings in presence of oPRL (ng/ml):.sup.+                Antibody*                                                                           Final*                                                                             1.000         0.125                                                added dilution                                                                           -Met.sup.14 hGH                                                                      +Met.sup.14 hGH                                                                      -Met.sup.14 hGH                                                                      +Met.sup.14 hGH.sup.                          __________________________________________________________________________    None  --   2.13 ± 0.01                                                                       1.23 ± 0.04.sup.                                                                  1.14 ± 0.03                                                                       0.42 + 0.05.sup.                              anti-hIge                                                                           1:100                                                                              2.11 ± 0.02                                                                       1.23 ± 0.01.sup.                                                                  1.16 ± 0.03                                                                       0.44 ± 0.02.sup.                           anti-hGH                                                                            1:100                                                                              2.11 ± 0.02                                                                       2.09 ± 0.06                                                                       1.19 + 0.03                                                                          1.14 ± 0.01                                anti-hGH                                                                            1:400                                                                              2.10 ± 0.02                                                                       1.92 ± 0.03.sup.                                                                  1.14 ± 0.01                                                                       0.98 ± 0.03.sup.                           anti-hGH                                                                            1:1600                                                                             2.11 ± 0.01                                                                       1.68 ± 0.01.sup.                                                                  1.11 ± 0.02                                                                       0.62 ± 0.02.sup.                           anti-hGH                                                                            1:6400                                                                             2.10 ± 0.02                                                                       1.46 + 0.01.sup.                                                                     1.12 ±  0.01                                                                      0.51 ± 0.02.sup.                           __________________________________________________________________________     *Ascitic fluids                                                               .sup.+ av ± SEM, n = 2, through 66 h.                                      .sup.  12 μg/ml                                                            .sup. Significantly different from the respective control without             Met.sup.14 hGH (p <0.001).                                               

                                      TABLE 3                                     __________________________________________________________________________    Effect of various concentrations of bPRL on the inhibition of the bPRL        stimulated                                                                    fatty acid synthesis by Met.sup.14 hGH in explants from bovine lactating      mammary gland                                                                            Fatty acid synthesis (dpm/mg/4 h) in the presence of various       Met.sup.14 hGH                                                                           concentrations of bPRL (μg/ml):*.sup.+                          Cow no.                                                                            (μg/ml)                                                                          0      0.2    1.0    2.0    20.0                                   __________________________________________________________________________    1    0     3814 ± 89                                                                         5064 ± 326                                                                        5809 ± 393                                                                        ND     ND                                          10    ND     3220 ± 93.sup.                                                                    4812 ± 258.sup.                                                                   ND     ND                                          50    3212 ± 88.sup.                                                                    3257 ± 259.sup.                                                                   2682 ± 119.sup.                                                                   ND     ND                                     2    0     476 ± 55                                                                          859 ± 47                                                                          1033 ± 57                                                                         ND     ND                                          10    ND     549 ± 47.sup.                                                                     598 ± 39.sup.                                                                     ND     ND                                          50    486 ± 45                                                                          525 ± 29.sup.                                                                     525 ± 57.sup.                                                                     ND     ND                                     3    0     822 ± 35                                                                          1496 ± 366                                                                        ND     ND     2080 ± 322                               20    793 ± 80                                                                          1061 ± 61                                                                         ND     ND     1300 ± 181.sup.                     4    0     2046 ± 241                                                                        2981 ± 124                                                                        ND     2979 ± 236                                                                        2280 ± 258                               20    1088 ± 148.sup.                                                                   1126 ± 61.sup.                                                                    ND     1496 ± 175.sup.                                                                   1696 ± 186                          5    0     449 ± 37                                                                          735 ± 50                                                                          ND     1150 ± 118                                                                        1048 ± 88                                1     441 ± 21                                                                          778 ± 56                                                                          ND     896 ± 50                                                                          881 ± 80                                 2.5   514 ± 12                                                                          612 ± 60                                                                          ND     994 ± 57                                                                          724 ± 47.sup.                            10    430 ± 12                                                                          431 ± 45.sup.                                                                     ND     743 ± 56.sup.                                                                     645 ± 58.sup.                       __________________________________________________________________________     *All treatments included insulin (1 μg/ml) and hydrocortisone (0.5         μg/ml)                                                                     .sup.+ av ± SEM, n = 6                                                     .sup. Significantly different from the respective treatment without Met       hGH: (p < 0.05)                                                               .sup. as above (p < 0.01)                                                     .sup. as above (p < 0.0001)                                              

Full or almost full displacement of the ligand could be achieved but theamounts of Met¹⁴ hGH required for 50% of displacement were respectively83- and 35-fold higher than those of hGH.

Discussion

Utilization of two separate bioassay systems enabled us to demonstrateclearly that the recombinant Met¹⁴ hGH inhibits hGH- and oPRL-stimulatedmitogenesis of Nb2 lymphoma cells and bPRL-stimulated fat synthesis inexplants of bovine lactating mammary gland. In the latter system Met¹⁴hGH also inhibited (cows no. 3-5) bPRL stimulated secretion ofα-lactalbumin (not shown), thus implying more general inhibition ofprolactic action. It should be noted that in both systems the inhibitiondid not result from impaired cell viability. No cytotoxic effect wasfound in Nb2 cells exposed to Met¹⁴ hGH. Glucose uptake in mammary glandexplants was not affected by various treatments. It should be noted thatin cows no. 1 and 4 Met¹⁴ hGH also inhibited basal activity. The reasonfor this is not clear but may be related to a much higher intrinsic fatsynthesis in these cows. Whether the latter results from carryover ofendogenous prolactic or PRL-stimulated activity is not clear.

Binding experiments revealed that Met¹⁴ hGH competes with ¹²⁵ I-hGHalthough its affinity is lower than that of hGH. These results implythat Met¹⁴ hGH may act as an antagonist, namely blocking the agonist atthe receptor or post-receptor level. The maximal rate of proliferationwas not changed by the inhibitor but the apparent affinity of eitheroPRL or hGH was decreased. Although the biological significance of thisconstant is not clear since it is a function of both binding andpost-binding events its value in the absence of Met¹⁴ hGH (3.85 pM forhGH and 2.95 pM for oPRL) is close to the value of 5.8 pM of ¹²⁵ I-hPRLrequired for half-maximal growth of the cells (7). It was found that inorder to achieve 50% growth inhibition 10³ -10⁴ molar excess of Met¹⁴hGH were needed, despite the fact that the affinity to bind to intactcells in only ˜85 fold lower. This may be related to the finding thatfull growth response may be achieved at 35% receptor occupancy (7).

Unlike in Nb2 cells, the competitive inhibition of bPRL stimulated fatsynthesis in explants is much less obvious. A trend showing thisdirection was seen in cow number 1 where the inhibition with 10 μg/ml ofMet¹⁴ gGH was significantly lower at 0.2 μg/ml, than at 1.0 μg/ml, andto a lesser degree in cow numbers 2, 4 and 5. In the latter case, 10μg/ml of Met¹⁴ hGH absolutely abolished PRL stimulation at the 0.2μg/ml, while at 2 and 20 μg bPRL/ml there was only partial inhibition.It should be remembered that the explants consist of primary culturesthat are much more variable to the absolute and relative hormonalstimulation than the Nb2 cells. The explant system seems also to be moresensitive, and significant inhibition was achieved in 0.5-50 Met¹⁴hGH/bPRL molar ratios. Although the apparent affinity constant for thebinding of hGH to the mammary gland receptors is 20-35 fold higher thanfor Met¹⁴ hGH we have previously demonstrated (9) that it is also20-fold higher than for bPRL thus making the binding constant of thelatter and the Met¹⁴ hGH quite comparable.

The finding that the preparation of Met¹⁴ hGH used in this investigationalso contained some low molecular weight degradation products raises apossibility that they, rather than the main 20K band, may be responsiblefor the inhibitory effect. The fact that monoclonal antibodies againsthGH abolished the inhibitory activity of Met¹⁴ hGH argues against thissuggestion. In addition, we have purified small amounts of Met¹⁴ hGH byimmunoaffinity chromatography on anti-hGH monoclonal antibody coupled toSepharose. The purified material gave a single 20K band on NaDodSO₄/polyacrylamide gel electrophoresis and retained its full inhibitoryactivity against oPRL-stimulated proliferation of Nb2 cells (data notshown).

To our knowledge this is the first report describing inhibition of hGHactivity by a modified hGH. Careful examination of the data compiled byPaladini, et al. (13) reveals that removal of the N-terminal portion ofhGH drastically reduced and in most cases absolutely abolished itslactogenic or somatotrophic activity. The importance of the N-terminalpart of oPRL for its in vivo mitogenic activity also has been reported(14). In view of these results and our present findings it seems thatthe N-terminal part of these hormones is obligatory for expression ofactivity. Its removal may cause inactivation or even conversion of anagonist to antagonist. It should also be remembered that in both systemsused in our experiments the hormonal effect was mediated throughlactogenic receptors. Whether similar inhibitory effect of Met¹⁴ hGH canbe found in somatotrophic receptor-mediated activity has yet to beinvestigated.

REFERENCES

1. Chadwick, S., Folley, S. J., & Forsyth, I. A. (1961) Lancet, 2,241-243.

2. Doneen, B. (1976) Gen. Comp. Endocrinol., 34, 34-42.

3. Kleinberg, D. L. & Todd, J. (1980) J. Clin. Endocrinol, Met. 51,1009-1013.

4. Gertler, A., Cohen, N. & Maoz, A. (1983) Mol. Cell. Endocrinol., 33,169-182.

5. Gout, P. W., Beer, C. T. & Nobel, P. R. (1980) Cancer Res., 40,2433-2446.

6. Tanaka, T., Shieu, R. P. C., Gout, P. W., Beer, C. T., Noble, R. L. &Friesen, H. G. (1980) J. Clin. Endocrinol. Met., 51, 1058-1063.

7. Shiu, R. P. C., Elsholtz, H. P., Tanaka, T., Friesen, H. G., Gout, P.W., Beer, C. T. & Noble, R. L. (1983) Endocrinology, 113, 159-165.

8. Shiu, R. P. C., (1979) Cancer Res., 39, 4381-4386.

9. Gertler, A., Ashkenazi, A. & Madar, Z. (1984) Mol. Cell. Endocrinol,34, 51-57.

10. Bundeson, P., Drake, R. G., Kelly, K., Worsley, I. G., Friesen, H.G. & Sehon, A. (1981) J. Clin Endocrinol Met., 51, 1472-1474.

11. Gertler, A., Weil, A. & Cohen, N. (1982) J. Dairy Res., 49, 387-398.

12. Gertler, A., Walker, A. & Friesen, H. G. (1985) Endocrinology (inpress).

13. Paladini, A. C., Pena, C. & Poskus, E. (1983) CRC, Critical Reviewsin Biochemistry, 15, 1-56. 14. Mittra, I. (1980) Biochem. Biophys. Res.Commun., 95, 1760-1767.

What is claimed is:
 1. A polypeptide which inhibits the activity ofnaturally-occurring human growth hormone (hGH) or naturally-occurringprolactin (PRL), said polypeptide having the sequence ofnaturally-occurring hGH from what the first thirteen amino acids havebeen deleted and which has the methionine which occurs at position 14 ofthe naturally-occurring polypeptide at the N-terminus.
 2. Apharmaceutical composition comprising an effective amount of thepolypeptide of claim 1 and a suitable carrier.
 3. A method of treatinghuman growth hormone (hGH) excess or prolactin (PRL) excess whichcomprises administering to a subject having such an excess an effectiveamount of the polypeptide of claim 1.